Procedure for the large-scale T-lymphocytes culture in a homogeneous system

ABSTRACT

The present invention refers to a procedure for the large-scale amplification of human lymphocytic cell lines for therapeutic use, consisting of a homogeneous culture system. The claimed invention also refers to the production of therapeutic doses of lymphocytic cells cultured homogeneously.

RELATED APPLICATIONS

This application is a U.S. National Phase under 35 U.S.C. 371 ofInternational patent application No.: PCT/EP2003/011024, filed Oct. 6,2003 designating the United States of America and published in Englishon Apr. 15, 2004 as WO 2004/031370, which claims the benefit of priorityof Italian Patent Application No.: MI2002A 002118, filed Oct. 4, 2002.

FIELD OF THE INVENTION

The field of the invention concerns the in vitro cell culture and thelarge-scale expansion of isolated human cells.

PRIOR ART

An approach to the anti-tumour therapy is based on the use of ex vivoisolated cell lines or cells endowed with cytotoxic activity.

In the early '90, a number of T-lymphocyte cell lines derived fromchildren with acute T-cell lymphoblastic leukemia, named TALL, wereisolated. They include T-cell lines TALL-104, TALL-107, TALL-103/2,described by O'Connor et al. (Blood, 1991, 77: 1534-1545) and by Cesanoand Santoli (In Vitro Cell. Dev. Biol., 1992, 28: 648-656). They exhibitsuch interesting characteristics that they are now-successfully used inthe treatment of tumours in animal models and in man (Cesano et al.,Blood 1991, 87:393-403; Cesano et al., Cancer Res., 1996, 56: 3021-3029,U.S. Pat. No. 5,272,082; U.S. Pat. No. 5,683,690; U.S. Pat. No.5,702,702).

TALL cell lines are endowed with cytotoxic, specifically anti-tumour,activity and are active against different types of tumours: the maincharacteristic of these cells is that they are MHC non-restricted(Cesano et al., J. Immunol., 1993, 151: 2943-2957) and, therefore, canbe administered to any patient, independently of the histocompatibilityantigens phenotype. Furthermore, unlike some types of cytotoxiclymphocytes, such as for example TIL and LAK, TALL cell lines do notneed, after in vivo administration, a concomitant treatment withlymphokines. This is a further advantage of said cells, since thesimultaneous administration of lymphokines has several drawbacks.

Moreover, TALL lymphocytes have been successfully tested in a variety oftumours. These are the reasons why they are considered an interestingtherapeutic alternative. However, the cell expansion systems used so farare limiting because the cells prepared for adoptive immunotherapyderive from cultures grown in single flasks, as described in Cesano etal., Cancer Res., 1996 56: 4444-4452 and Visonneau et al., Clin. CancerRes., 1997, 3: 1789-1797, although large-scale cell cultures apparatuseslike those used in the preparation of monoclonal antibodies andrecombinant proteins have long been utilised. Therefore, a large-scaleculture system suitable for cells of this type is highly desirable.

SUMMARY

It is an object of the present invention to provide a process for theTALL lymphocytes large-scale expansion and growth based on the use of ahomogeneous culture system. The expression “large-scale amount of TALLlymphocytes” refers to 1×10⁹ cells at least.

In particular, the fermentor or homogeneous system used in the claimedprocess is a cell-factory, preferably consisting of a stack of 10chambers. The lymphocytes that may be expanded according to the presentprocedure are selected from the group consisting of TALL-104, TALL-107,TALL-103/2, optionally genetically modified.

According to a further embodiment the invention concerns a process ofcell—sampling said process based on the sealing of the filling collet ofthe bag which creates at least a sampling chamber containing a number ofcells sufficient for sampling.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart illustrating the glucose levels and the cellulardensity of TALL cells grown in flask.

The following parameters were determined in 15 flasks of TALL: cellsnumber/ml (-♦-) and glucose levels (full bar).

DETAILED DESCRIPTION OF THE INVENTION

The invention refers to a procedure for the large-scale expansion ofTALL lymphocytes, wherein at least 1×10⁹ cells are cultured in ahomogeneous system preferably consisting of a single fermentation unit.

TALL (T-cell acute lymphoblastic leukemia) lymphocytes are cytotoxicT-lymphocyte lines derived from a pediatric patient with lymphoblastoidleukemia and include TALL-104, TALL-107, TALL-103/2 lines, as describedin O'Connor et al., Blood, 1991, 77, 1534:1545, and Cesano and Santoli,In Vitro Cell. Dev. Biol., 1992, 28: 648-656). The preferred cell lineis TALL-104. TALL cells may also be modified genetically.

They have so far been exclusively amplified in single flasks up to anobtainable maximum of approx. 1×10⁸ cells/T175 flask.

Therefore, the preparation of bags containing therapeutically effectivequantities of cells comprised in the range from 10⁵ to 10¹² cells,preferably from 1×10⁷ to 1×10¹⁰ and still more preferably from 1×10⁸ to2.5×10⁹ involved, until now, the simultaneous amplification of a largenumber of single flasks.

According to the definitions of the invention the growth in singleflasks represents a heterogeneous culture system since each flaskrepresents a different culture microenvironment. Consequently, and inaccordance with FDA's guidelines: “Guidance for Industry: Guidance forhuman somatic cell therapy and gene therapy” (CBER, March 1998, PointIII) of the US Department of Health—which recommends a separate controlof each mixture of cells prepared in an independent system—the cellsderived from each single flask according to the prior art methodsrepresent different lots and, therefore, are to be controlledindependently. It follows that the development of a homogeneous culturesystem of cells for therapeutic use, and especially of TALL cells,represents an enormous advantage also as far as lot controls areconcerned.

Furthermore, the amplification in a heterogeneous system, which consistsof a number of single flasks, may bring about high risks ofcontamination due to the repeated handling operations to be performed bythe operator.

TALL cells usually grow in suspension but, surprisingly, they cannot beamplified or expanded in known systems for the industrial scale-up ofcells of this type, e.g. spinner flask or miniPERM. Therefore, when theamount of cells to be produced exceeds 10⁹, such as for example for theproduction of therapeutic doses of 2.5×10⁹ cells at least, the steps ofcell—expansion become extremely complex since the largest flasks (T175)commercially available allow the obtainment of 1.5-2×10⁸ TALL cells atmaximum.

The applicant has surprisingly found that said cells can be efficientlygrown and expanded in a homogeneous system such as a cell-factory whichis normally used for anchorage-dependent cells. Conversely, the spinnerflask or other fermentors conventionally used for the large-scale growthof cells in suspension, e.g. hybridoma cells exhibiting growthcharacteristics similar to TALL cells, result to be inappropriate.

The expression “large-scale production” means the production of at least1×10⁹ TALL cells in a homogeneous system.

In the procedure of the invention, the data referred to the number ofcells have a tolerance of approx. 5%, which represents the possibleerror in the cell count determined in Burker's chamber. For example, theindication of 1×10⁶ cells actually refers to a number of cells of0.95×10⁶ to 1.05×10⁶. The error of measurement is variable and dependson the method of measurement adopted.

According to the procedure of the invention, the TALL expansion in acell-factory is preferably preceded by a pre-expansion consisting in aseries of volumetric expansions in the same flask (wherein for flask isintended a cell culture container) by means of successive additions offresh complete medium to the culture and by transfer passages of thewhole culture into higher-volume flasks and finally into thehighest-volume and largest-surface flasks commercially available such asthe T175 flasks.

According to a preferred feature of the present invention, thepre-expansion is performed until obtaining about 3-4×10⁸ total cells ofwhich about 2-2.5×10⁸ cells are used for the inoculation into eachcell-factory and about 1-1.5×10⁸ cells are in parallel maintained InT175 culture flasks.

Splitting of the cell culture, performed to bring back the cellulardensity values to the optimal inoculum values, i.e. 0.7 to 1×10⁸cells/ml, is referred to herein as “passage”. At this density the cellsrapidly reach a density of approx. 2.5×10⁹ cells/ml.

Pre-amplification is preferably carried out in a complete medium, morepreferably IMDM, containing 2 mM glutamine, foetal bovine serum (FBS) inconcentration from 2 to 20%, preferably 5%, and cytokines, preferablyinterleukins, and more preferably IL-2 or IL-15. IL-2 is preferablyadded in an amount of 100 IU/ml every 48-72 hrs. In the homogeneoussystem, the medium of the amplification phase is the same as that of thepre-amplification phase, but foetal bovine serum is replaced, at leastpartially, by human serum AB. Said replacement can also be performedbefore cell—transfer to the homogeneous cell culture system, for examplein the last passages of the pre-amplification phase. IMDM may bereplaced by other culture media, such as for example RPMI, Ham's-F12,etc. The medium is preferably antibiotic-free.

A medium preferably IMDM containing 2 mM glutamine but antibiotic-freeis referred to herein as “complete medium”; it may be supplemented withFBS or human serum.

Interleukins, preferably IL-2 or IL-15, are added to the cell culturemedium every 48 to 72 hrs, in a final concentration of 50 to 150 IU/ml,more preferably of 100 IU/ml.

The cells are incubated at 37° C. and in an air mixture comprisingpreferably from 5 to 12% CO₂ preferably 10% CO₂, preferably 10% CO₂. Allpassages envisaging cells or culture media handling operations areperformed in sterile conditions, e.g. in a vertical laminar flowBiohazard hood (class 100).

According to a particularly preferred embodiment, the pre-amplificationculture is started from a frozen MCB (Master Cell Bank) culture tube orvial, stored in liquid nitrogen and containing 1×10⁷ to 2×10⁷cells/tube/ml which is thawed out in a thermostatic bath at 37° C.

The vial content is added, in a laminar flow hood, with an 8 to 10 timeshigher volume of cold thawing solution (complete IMDM comprising 20%FBS). The cells are centrifuged (at 1500 rpm for 10 min). Once thesupernatant has been aspirated off, the cell pellet is added with 10 mlcomplete medium and resuspended cells are caused to pass into a T25flask. IL-2, diluted with complete medium, is added to a finalconcentration of about 100 IU/ml. When the cell density obtained is suchthat the cells occupy the whole flask surface, as observed under aninverted microscope, the cell culture is expanded in twice the mediumvolume (and amplified in two T25 flasks). For this purpose, an equalvolume of fresh medium is added and the culture is split into two equalflasks to restore the optimal cellular density (inoculum cellulardensity), which generally ranges from 0.7 to 1×10⁶ cells/ml, and whereinthe volume of each flask corresponds to the initial volume.

To maintain an optimal gas exchange in the culture medium, the optimalcell culture medium volume in a T25 flask is comprised from 7 to 12 ml,preferably 10 ml; of a T75 flask it is from 20 to 60 ml, preferably 40ml; of a T175 flask it is from 40 to 200 ml. The above volumes areapproximate and are referred to optimal cellular density conditions.

After thawing, the optimal cellular density is usually reached after 3to 7 days, preferably after 5 days. After approx. 3 days, the cells arecaused to pass from 2 T25 flasks into 2 T75 flasks; the cells left inthe flask, if any, are harvested; IL-2 is added to the fresh mediumaccording to the above indicated concentration.

After approx. 3 days, the cells are caused to pass from 2 T75 flasksinto 2 T175 flasks. The cells left in T75 flasks, if any, are harvestedwith washing in complete medium to reach a final volume of 40 ml andadded with IL-2 in proportion. After 2-3 days, the cells are caused topass from 2 TI 75 flasks into 4 20 ml T175 flasks, whereto the samevolume of complete medium and IL-2 in proportion are Immediately added.After 2 days, the cells are caused to pass from 4 T175 flasks into 8T175 flasks. Each T175 flask is added with approx. 20 ml complete mediumand IL-2 in proportion. After approx. 2 days, the 8 T175 flasks areadded with approx. 40 ml complete medium comprising human serum inconcentration ranging from 2% to 10%, preferably from 4% to 6%, morepreferably of 5%, and IL-2 in proportion. After 2 days, the 8 T175flasks are added with approx. 80 ml complete medium containing IL-2 inproportion.

The final harvest volume of T175 flask usually ranges from 140 to 180ml. The pre-amplification phase is completed after approx. 15 days witha number of cells ranging from 0.9×10⁹ to 1.1×10⁹ (which value generallycorresponds to 8 T175 flasks, each containing 160 ml cell suspension) ina medium containing human serum in a final concentration comprised from2% to 5%, preferably from 3% to 4%, and optionally foetal bovine serum(FBS) in a final concentration from 0.0 to 5%, preferably from 1.5% to3%.

The passage from the medium containing FBS to the medium containinghuman serum preferably occurs during the two last passages of thepre-amplification phase in flask, preferably T175, preferably bysuccessive dilutions of the culture containing FBS with fresh mediumcontaining human serum.

Inoculum into cell-factory is performed with a number of cells rangingfrom 1.5 to 2.5×10⁷/chamber in a volume ranging from ⅙ to 1/10,preferably ⅛, of the cell-factory final volume capacity. For theinoculum into a 10-chamber cell-factory having a final capacity of 2litres, the inoculum is performed with 1.5 to 2.5×10⁸ in a medium volumefrom 200 to 330 ml, preferably 230 to 270 ml, immediately added with thesame volume of fresh complete medium containing 10% max, preferably 5%,human serum and cytokins, preferably interleukins, more preferably IL-2or IL-15, still more preferably IL-2, in a final concentration from 80to 120 IU/ml, preferably 100 IU/ml.

Every 3-5 days, preferably every 4 days, during which time the cellsgenerally duplicate, a volume of complete medium corresponding to thatcontained in the cell-factory is added to continue the cell expansionand growth up to a max final volume of 2 litres/10-chamber cell-factoryand to a number of cells of 1.5 to 2.5×10⁹. The amplification of TALLcells in cell-factory requires the addition of fresh medium containinghuman serum in an amount of 10% max, preferably from 3 to 7%, morepreferably from 4 to 6%, still more preferably of 5%. Therefore, in theamplification phase in the homogeneous culture system according to theinvention, preferably no or little foetal bovine serum is used. Tracesof FBS, if any, which maybe present at the end of the process of theinvention, are the result of successive dilutions of medium containingFBS with the medium containing human serum.

Briefly, according to a procedure general scheme, both in thepre-amplification phase in flask and in the expansion phase in thehomogeneous system, the cellular density, at the inoculum is never below0.7×10⁶ cells/ml (which value corresponds to the value obtainable bysplitting the cell culture into 2 or into 3 every 48-72 hrs) and ispreferably 0.75×10⁶/ml. Conversely, in the final growth phase, shortlybefore cells harvesting, it never exceeds 2×10⁶ and is preferably1.5×10⁶/ml.

The volumes and the number of cells according to the process of theinvention—in which the inoculum is performed into a 40-chambercell-factory with a medium max capacity of 8 litres and capable ofsupplying a total amount of cells grown in a single homogeneous systemof approx. 8-10×10⁹, have been proportionally calculated.

According to the process of the present invention, at least one bagcontaining the highest therapeutic dose of TALL cells, i.e. 2.5×10⁹cells (bags containing 1×10⁸ to 2.5×10⁹ cells), can be produced by ahomogeneous culture system. Therefore, according to a further embodimentof the invention, the process embraces also the preparation of frozenbags of TALL lymphocytes in an amount of at least 1×10⁹, characterizedby the fact said that TALL cells are expanded in a homogeneous culturesystem. The bags used have a variable volume and, therefore, containdifferent therapeutic doses of cells. Bags for freezing and for infusionare preferably used, more preferably Baxter Cryocyte's bags.

To prepare bags for therapeutic use, cells from the cell-factory at theharvest, are placed in a 50 ml sterile tube and irradiated, according tomethods known to the art, in a beta particle accelerator (betatron). Themethod has been validated so as to supply the same amount of particlesas that supplied by a traditional source, such as Cs137. The methodusing the betatron has the advantage that it is not radioactive andbrings about a uniform irradiation of the solution. Once irradiated, thecells are centrifuged (at 1500 rpm for 10 min). At this point, a sampleof cells is withdrawn for qualitative tests.

The irradiated cells together with the final suspension components andthe sterile bags are placed in a laminar flow hood. The cells areresuspended in the freezing solution consisting of Rimso 50 (50% DMSO)(20%) and of 5% human albumin (80%) in 8 to 30 ml medium, morepreferably in 10 to 25 ml complete medium containing TALL cell doses inthe range of 1×10⁸ to 2.5×10⁹. Preferably, the cellular density in thebag ranges from 10⁶ to 10⁸ cells/ml. The irradiated cells may be storedat 4° C. for no more than 24 hrs before freezing.

The cell concentration is evaluated by count, under a microscope, inBurker's chamber, or by other methods known to those skilled in the art.Bags are filled in sterile conditions.

Once the bags have been filled, they are sealed, e.g. by hot sealing,transversally to the bag filling collet, in two, preferably threepoints, to create one or preferably two chambers which contain cells,and wherein such cells are called “authentic samples” for the purpose ofthe present invention. The cells aliquots contained in the two bagcollet portions are separate but derive from the same culture batch.

According to a preferred embodiment of the invention, the volume of thecells suspension contained in the collet chambers ranges from 0.1 to 1ml, preferably 0.3 ml, and the number of cells/chamber is sufficient forat least a series of appropriate quality and/or sterility controls. Thecells of the chamber/s corresponding to the authentic samples, may beeasily withdrawn without opening the whole bag.

Said chambers containing the “authentic samples”, the method of sealingthe bag collet in one or more points and the method of formation of thebag authentic samples are further objects of the present invention.

The bags for cryopreservation and infusion are frozen at −80° C.

It is a further object of the present invention to provide a process forthe preparation of frozen bags of TALL lymphocytes in an amount of atleast 1×10⁹ cells, wherein said amount derives from a single homogeneousculture system.

The quality controls on cells amplified according to the process of theinvention may be performed before freezing upon cells harvesting fromthe homogeneous culture system as well as after freezing, and are meantfor checking the stability of the properties, such as the percentamounts of immunologic markers and the biological activity of the TALLcells finished product. The cells produced according to the inventionare stable also after freezing.

The quality controls preferably comprise the following measurementsperformed according to methods known to the art:

-   -   viability, preferably determined by the Trypan Blue exclusion        test; it must be 80% min;    -   biological activity, preferably determined by a cytotoxicity        test (adenylate-kinase measurement), although alternative tests        may also be used; it must be higher than a 60% lysis of target        cells, K562, in a 10/1 ratio;    -   endotoxin levels, preferably determined by the Lymulus Amebocyte        Lysate colourimetric test; they must be ≦0.5 EU/mL;    -   immunologic markers phenotype, preferably determined by        immunofluorescence (FACS), which must give values of at least        90% for the markers known as CD3⁺, CD8⁺, CD56⁺;    -   proliferation, preferably performed by the ³H-TdR incorporation        test: the proliferation measured after 72 hrs at least must be        higher than or equal to twice the background value.

According to the method of the present invention, the parameter measuredsuch as the percent of immunologic markers, the biological activity, andthe activity of metabolic markers, such as for example the glucose levelof the culture medium, are less variable in cell grown in the homogenoussystem than those measured on cells grown in a heterogeneous culturesystem.

In fact, in the cell-factory homogeneous culture system, the percentageof immunologic markers expressed on TALL cells is at least 90%, morepreferably at least 95% more preferably at least 98%, for CD3⁺ andCD56⁺; and at least 90%, more preferably at least 93% for CD8⁺, whereasit is lower for cells grown in flask. The value of CD56⁺ markerexpression which results preferably at least 95% min, preferably atleast 97%, is higher in the process of the invention than in cells grownin the heterogeneous flask system. Also the biological activitydetermined by the cytotoxicity test on target cells, which are usuallypreferably K562, is higher than the acceptable limit and is alwayshigher than 70% compared to the control which consists of an appropriatenumber of cells where lysis has been completely induced. According to afurther embodiment the invention comprises the TALL lymphocytes,preferably TALL 104, obtainable according to the process of theinvention.

Also the glucose levels measured proves that the metabolic conditions ofthe cells are more homogeneous in cells grown in cell-factory accordingto the process of the invention with respect to those grown in flasks(FIG. 1): in fact, while the measurements in flask give glucoseconcentrations ranging from 300 to 400 mg/dl, the measurements incell-factory give values ranging from 350 to 380 mg/dl. According to asimplified embodiment, the process of the invention comprises any TALLcell culture pre-amplification phase performed according to methodsknown to the art, comprising an inoculum in cell-factory of 2×10⁷cells/chamber, in an initial volume of 1/10 to ⅙ of the cell-factoryfinal volume, and a cell-amplification phase in cell-factory, preferablyin a 10-chamber cell-factory (with a final capacity of approx. 2 L),with a total inoculum of approximately 2×10⁸ cells in about 250 ml.

Under the conditions adopted in the present procedure, in thelarge-scale expansion in cell-factory, the ratio of minimum inoculumdensity to the maximum recovery density at the end of the expansioncycle is optimised. In fact, according to the method of the presentinvention, at the end of the expansion cycle the number of cells isapprox. ten times greater (from 1.5 to 2.5×10⁸ cells to 1.5 to 2.5×10⁹cells), whereas the volume is only 8 times higher. The furtheradvantages of the homogeneous culture system are: 1) elimination of thecell growth heterogeneity conditions, 2) reduced number of handlingoperations and, consequently, reduced possibility of contamination, 3)reduced man-hours and personnel costs.

A further advantage of the procedure according to the invention, whichderives from a reduced contamination risk in respect of culture systemsin multiple flasks, is the use of an antibiotic-free culture medium.

A still further advantage of the procedure according to the invention isthe use of a human serum concentration preferably of 10% max. Therefore,according to the procedure of the invention, a satisfactory cellamplification is obtained with a human serum concentration preferablyranging from 4 to 6%.

Therefore, by performing the expansion phase in cell-factory, the wholeprocedure for the preparation of the bag for the therapeutic use of TALLis optimised and is suitable for large-scale application.

The advantages of the TALL expansion method according to the presentinvention may be summarized as follows: i) the quality controls arelimited to a single sampling unit which corresponds to a singleproduction unit (cell-factory); conversely, the possibility ofcontamination increases when the expansion Is obtained by amplificationin a much greater number of single fermentation units, worked atdifferent times and, consequently, liable to increased risks ofcontamination; ii) the use of a limited number of bioreactors results inca. 30% time-saving and a 25% cost reduction.

It is to be noted that, according to the methods already known to theart for this type of cells, the amount of 2.5×10⁹ cells, correspondingto approx. 0.25 10⁸ cell bags or to 1 2.5×10⁹ cell bag was obtained with35 T175 and with a volume of complete medium of 2.8 L. It follows that,by the method according to the present invention, the saving in rawmaterials (human serum, medium, cytokines) is as high as approx. 30%.

A further advantage is obtained on the number of controls conducted onthe final lots, which consist of 1×10⁸ cells max in the heterogeneoussystem known to the art and of a 10 times greater number in the lotproduced by the homogeneous system described in the present invention.In practice, this means that the number of controls is 10 times lower.

EXPERIMENTAL EXAMPLES 1. Materials

Flasks: Falcon, Beckton Dikinson;

cell factory: cat. No. 164327 or 170009, Nunc A/S, Denmark;

www.nuncbrand.com;

culture medium: Iscove's Modified Dulbecco Medium, Biowhittaker 12-722,supplemented with glutamine;

CM (Complete Medium) containing glutamine and serum;

foetal bovine serum: Biowhittaker, USA;

human serum, type AB, Biowhittaker;

IL-2 Proleukin 1, Chiron;

saline solution: phosphate buffered saline, Biowhittaker;

human albumin (5%), Farma Biagini;

RIMSO 50, Baxter;

bags for cryopreservation and infusion, Cryocyte Baxter.

2. Methods

All cell cultures were performed in a sterile environment, inBiohazard's hood. At every addition or withdrawal, inlet spouts weredisinfected with isopropanol or with the flame.

2.1. Medium Preparation

Serum lots were uncomplemented in a thermostatic bath at 56° C. for 1 h.Uncomplemented serum bottles were kept at 4° C. and used within onemonth from the date of uncomplementation.

2.2. Complete Medium for the Pre-Amplification Phase

A 500 ml IMDM bottle was added with uncomplemented FBS (50 ml); thebottle was identified with the lot. The complete medium was stored at 4°C. and heated to room temperature before use. The complete medium wasused within one month from the date of preparation.

2.3. Complete Medium for Scale Up in Cell-Factory

A 500 ml IMDM bottle was added with 25 ml uncomplemented human serum AB.The medium was stored at 4° C. and heated to room temperature beforeuse. The complete medium was used within one month from the date ofpreparation.

2.4. IL-2 Preparation

Interleukin (18×10⁶ IU/vial) was resuspended in 20 ml PBS to obtainapprox. 9×10⁵ IU/ml. The resulting solution was filtered through 0.22 μmfilters and the vials were dispensed in 1 ml aliquots. The aliquots werestored at −80° C.

This solution (1 ml) was diluted with complete medium, corresponding tothe medium of point 2.2. for the pre-amplification phase, then with amedium corresponding to point 2.3. for the amplification phase, toobtain a 10⁴ IU/ml solution. Said solution was added to the cellsuspension, by diluting same (1/100) with the culture medium.

2.5. Process Controls

Bioburden: The microbial contamination was determined on the completemedium used to grow TALL cell lines (IMDM+supplemented serum). Theresulting medium was filtered through a membrane with 0.45 μm pores, thefilter was removed by a sterile lancet and placed in an appropriatemedium for the visualisation of the micro-organisms contamination (themedium used was Tryptic soy agar). The filter was laid on the plate. Thepresence of moulds was detected after incubation at 20-25° C. for 3 daysat least and the presence of bacteria was detected after furtherincubation at 30-35° C. for at least 3 days. Plates were visuallyinspected to check the growth of micro-organisms (100 CFU AT maximum).

Microbiologic monitoring: The hoods used for TALL processing werecontrolled to check whether micro-organisms, If any, were present duringthe procedure. While the operator was working on the cells (pouring,bags filling), a Petri dish with TSB agar was exposed in the hood,covered upon process completion, and incubated at 20-25° C. for 3 daysat least to detect the presence of moulds and at 30-35° C. for 3 days atleast to detect the presence of bacteria. Micro-organisms were foundduring sampling in two cases of flasks handling operations, whereas nocontamination was detected in the cell-factories handling operations.

Example 1 Laboratory-Scale Thawing and Amplification

A thawing solution consisting of IMDM medium supplemented with 20% FBSwas prepared and kept at 4° C. before use.

An ampoule containing frozen cells was taken from the nitrogen drum,thawed appropriately, and diluted (1:10) with the thawing medium. Thecells were centrifuged at a low rate for 10′. The pellet was resuspendedin 10 ml complete medium, caused to pass into a T25 flask, and addedwith IL-2 up to a concentration of 100 IU/ml.

The cells were incubated at 37° C. for 5 days in an environmentcontaining 10% CO₂.

After a 5-day incubation, the cells were split (1:2) in a fresh mediumcontaining the same concentration of interleukin 2 and allowed to growto confluence, which was maintained for 2 days. The cells were gentlyremoved from the flask and caused to pass into a T75 flask containing afinal medium volume of 20 ml and the same IL-2 concentration (100IU/ml). Once confluence had been reached in approx. 2-3 days, the cellswere allowed to stand for 2 further days and then amplified in a T175flask with a final medium volume of 40 ml and IL-2 (100 IU/ml). Two dayslater, further 40 ml of interleukin complete medium was added; the cellswere allowed to grow for 2 further days and split (1:2) into 2 otherT175 flasks in a final volume of 80 ml/T175.

The cells morphology and density were examined under a microscope. Thenthe cells were brought to a final volume of 150 ml/flask in 5% completemedium (IMDM containing human serum AB) and added with IL-2 inproportion.

The cells were split (1:2) into the same number of T175 flasks in afinal volume of 150 ml.

Example 2 TALL Cells Dynamic Growth in miniPERM Fermentor and SpinnerFlask

The cells were thawed, cultured in T25 and amplified up to a number of7×10⁵ cells/ml for inoculation into 0.5 L spinner flask or in miniPERM.

Some measurements meant to set up the growth parameters were performedin flask T75: the cells were split when they reached a concentration ofat least 1×10⁸/ml. To maintain a high cell viability (90% min) and ahigher cell splitting rate, the cells dilution was never below 7×10⁵cells/ml and the cells growth never exceeded 2×10⁶/ml. At the max cellconcentration, the glucose level was approx. 320-380 mg/ml. In T175, in80 ml final volume, a concentration of 1.25×10⁶ cells/ml, correspondingto a total of approx. 100×10⁶ cells/flask, was obtained.

It was assessed that, for a higher viability during freezing/thawing,the minimum number of cells/vial had to be higher than 1.2×10⁷. It wasalso observed that cells frozen in the logarithmic growth phase, ontheir turn reached the logarithmic growth phase after thawing, within9-10 days, whereas the cells frozen in other growth cycle phasesexhibited a higher time lag (10-12 days).

Spinner flask. The 0.5 L spinner flask had 300 ml working volume.Inoculation was performed with 1.5×10⁷ cells; spinning was set at 3 rpm;cell viability was measured 48 hrs later and was found to equal 50%. 120hrs later, all cells were dead.

Furthermore, 3 amplification tests were performed in MiniPERM (abioreactor with a high surface in respect of its volume, ideal for theexpansion of cells in suspension, such as hybridoma cells) using 10% CO₂and adopting the following specific conditions:

-   -   inoculation with 7×10⁷ cells in 30 ml at 10 rpm. The cell        viability started decreasing already after 48 hrs and all cells        were dead after 4 days. In the second test, the spinning rate        was decreased to 5 rpm. On the third day of growth, the cell        viability was 52%; on the 6th day all cells were dead. In the        third test, the inoculation was decreased to 3×10⁷ cells and the        spinning rate was decreased to 4 rpm. Although all cells had        died off within the 5th culture day, on the 2nd day the cell        viability was still fairly high (80%); this suggested how        important spinning is to cells survival. Both preliminary        fermentation tests had demonstrated that T-ALL cells could not        grow in the traditional large-scale growth conditions commonly        used for cells in suspension or anchorage-independent.

Example 3 Large-Scale TALL Amplification in Cell-Factory

A cell suspension (250 ml), obtained from the growth in T175 as perExample 1 and containing approx. 0.8×10⁶ cells/ml, were inoculated intoa 10-chamber cell-factory; the same amount of complete medium was added.Four days later, complete medium was added (500 ml×3 times) up to atotal volume of 2 L.

The cell-factory was emptied into 250 ml sterile vials—which werecentrifuged—and washed with sterile PBS; the cells were recovered invials and centrifuged again. Eight days after inoculation, the cellsrecovered from a cell-factory were approx. 2.35×10⁹.

In a typical production cycle in a 10-chamber cell-factory, 72inoculations and amplifications from a single MCB (Master Cell Bank)frozen vial were performed. Total duration: 120 days, total cellculture: 144 litres. The cells obtained were approx. 1.44×10¹¹.

Table 1 shows the data obtained from a fermentation in a 10-chambercell-factory and in a 40-chamber cell-factory, respectively.

TABLE 1 Yields obtained in a 10-chamber cell-factory and in a 40-chambercell-factory Cell factory 10 Cell factory 40 (2 liters) (8 liters) No.inoculations 72 31 Days 120 120 Volumes (liters) 144 248 No. of cells1.44 × 10¹¹ 2.48 × 10¹¹

The cell-factory cells content was combined in a 50 ml sterile vial andirradiated, as known, in a beta particle accelerator (betatron). Themethod had been validated so as to supply the same amount of particlesas that supplied by a traditional source, such as Cs137. The methodusing the betatron has the advantage that it is not radioactive andbrings about a uniform irradiation of the solution.

Bags preparation. Once irradiated, the cells were centrifuged (at 1500rpm for 10 min). A sample of cells was withdrawn for qualitative testsand stored at 4° C. before freezing.

The irradiated cells, together with the final suspension components andthe sterile bags, were placed in a laminar flow hood. The cells wereresuspended in the freezing solution, which consisted of Rimso 50 (50%DMSO) (20%) and 5% human albumin (80%), depending on the amount of cellsnecessary for a therapeutic dose. The cells were counted under amicroscope in Burker's chamber and the bags were filled with theappropriate volume in sterile conditions. Once the bags had been filled,the filling collet was sealed in three points; each bag thus formed anauthentic sample.

The cell-factory>irradiation>bags cycle was repeated a number of timessufficient to obtain the number of cells required by the lot.

Sterility Controls

Bioburden sterility controls, microbiological monitoring and mycoplasmamonitoring were continuously performed on media and hoods.

Example 4 Controls on the Finished Product and Comparison BetweenLaboratory-Scale and Large-Scale Growth

Measurement of glucose levels: the glucose levels were measured on asample of 15 flasks (out of the 35 flasks corresponding to the amount ofcells in a cell-factory) upon cells harvesting for bag preparation. Asshown in FIG. 1, the glucose concentration and the number of cells arehighly variable; conversely, in a 10-chamber cell-factory, uponharvesting (after 6-10 days) glucose shows one value (approx. 380 mg/dl)and the cells have one density (2×10⁹/ml). The different glucose levelsand the different cell concentrations found in flask are indicative ofnon-homogeneous metabolic conditions: in fact, high glucose levels inthe medium correlate with a low metabolic activity of the cell culture,whereas low glucose levels correspond to a high metabolic activity ofthe cell culture.

Final Product Quality Control

The controls listed in Table 2 were performed on irradiated cells. Theacceptability limits of the values are reported in the column on theright.

TABLE 2 Assay Method Limits Viability Trypan Blue >80% exclusion testBiological Cytotoxicity test >60% lysis of activity target cells K562 ina 10/1 ratio Endotoxins Lymulus Amebocyte ≦0.5 EU/mL Lysatecolourimetric test Phenotype Immunofluorescence ≧90% CD3⁺, (FACS) CD8⁺,CD56⁺ Proliferation ³H-TdR incorporation ≦twice the background after4-day incubationViability Test

The determination was performed by diluting 100 μl cell suspensionbefore bagging with a 100 μl Trypan Blue solution. After an appropriatedilution, a control was performed under a microscope by counting thedyed cells in Burker's chamber.

Biological Activity

The determination of cytotoxicity was performed on the cell suspension,before bagging, with a kit Toxilight™ (BioWhittaker LT07-217). Themethod is based on the measurement of adenylate-kinase bybioluminescence. Adenylate-kinase is released from the cells when theylose the membrane integrity and converts the substrate ADP into ATP inthe presence of Mg⁺⁺, thus allowing its measurement. The determinationwas performed by plating 10⁵ target cells, K562, onto a many-wellmicroplate; cells prepared according to the method were added, inamounts of 10⁶, 5×10⁵ and 2.5×10⁵, to obtain T/K (or effector/target)ratios equal to 10.5 and 2.5, respectively.

Once the target cells were incubated overnight, the % lysis of K562produced by cells prepared according to the method of the presentinvention, was always ≧70% of the maximum consisting of a sample of 10⁵K562 cells lysed by ultrasounds.

Endotoxins

The endotoxins content was determined on a sample with collets preparedas mentioned above, at neutral pH, using a kit for the chromogenic LALtest and applying the method described in European Pharmacopoeia, 4^(th)Edition, 2002, pp. 140-145.

Phenotype

The phenotype determination by measurement of the cell markers beforebagging was performed by immunofluorescence (FACS) with appropriateimmunologic markers (CD3⁺, CD8⁺, CD56⁺) available from Molmed's QualityControl Labs.

TABLE 3 Phenotypic markers of T-ALL cells grown in flask and incell-factory upon cells harvesting Amplification CD3 CD8 CD56Cytotox.act. ³H thymidine Flask  >90%  >90%  >90% — ≦twice thebackground after 4-day incubation Cell-factory avg  >98%  >93% ≧97% ≧70%≦twice the background after 4-day incubation Lot 1 pre  99% 90 98 post 99% — 99 Lot 2 pre 99 98 99 post 99 93 99 Lot 3 pre 99 97 92 post 99 9495 pre: pre-irradiation; post: post-irradiation; avg: average.Proliferation Test

The absence of T-ALL proliferation was determined by measuring theincorporation of tritiated thymidine. A sample of the cell suspensiontaken before bagging was incubated in microplates at differentconcentrations in complete medium (IMDM+serum+IL-2) for 4 days. ³H-TdR(2 μCi/ml, 50 μl/well) was added. After 3-hr incubation at 37° C., theradioactivity was measured. The cpm referred to TALL cells must be≦twice the background.

Table 3 shows the data concerning the proliferation of a cell batchgrown in flask and In cell factory, respectively.

Table 4 reports the data concerning the measurement (on differentpreparations) of the presence of immunologic markers, of cell viability,sterility and contamination by mycoplasma, if any, after cultureirradiation.

TABLE 4 Measurement of markers on TALL cells after irradiation No. ofCytofluorimetry ³H Thymidine incorp. samples cells CD3 CD8 CD56 samplebackground Sterility myco 2 2.5 × 10⁹ 99.59 94.8 92.51 24 27 OK negative6 2.5 × 10⁹ 99.8 91.34 97.17 33 27 OK negative 8 2.5 × 10⁹ 99.88 9397.84 37 26 OK negative 18  1 × 10⁸ 99.59 94.8 92.51 24 27 OK negativeNo. of Cytofluorimetry Thymidine incorp. samples cells CD3 CD8 CD56sample background Sterility myco 1 2.5 × 10⁹ 99.54 94.63 95.83 23 24 OKnegative 1 2.5 × 10⁹ 99.44 96.24 98.51 37 26 OK negative 8  1 × 10⁸99.54 93.36 99.79 34 25 OK negative 7 0.5 × 10⁹ 99.8 92.3 97.05 82 60 OKnegative 11 0.5 × 10⁹ 99.54 93.36 99.79 29 29 OK negative

1. A process for amplifying TALL-104 lymphocytes in a homogeneousculture system within a multi-chamber stack comprising: adding into themulti-chamber stack an inoculum of at least 0.7×10⁶ TALL-104 cells/ml inan initial volume from 1/10 to ⅙ of the multi-chamber stack volumecapacity and the same volume of fresh antibiotic-free complete medium,wherein said medium comprises interleukin-2 (IL-2); amplifying the cellnumber by adding a volume of said complete medium corresponding to thevolume contained in the multi-chamber stack every 3-5 days; andharvesting at least 1×10⁹ TALL-104 cells grown in homogeneousconditions.
 2. The process as claimed in claim 1, wherein said processfor amplifying TALL-104 lymphocytes is preceded by a process ofpre-expansion in a flask until obtaining a number of cells in an amountfrom 3×10⁸ to 4×10⁸.
 3. The process as claimed in claim 1, wherein thecellular density of the inoculum is 0.75×10⁶ cells/ml and, at theharvesting step, the density is lower than 2×10⁶ cells/ml.
 4. Theprocess as claimed in claim 1, wherein the multi-chamber stack is a10-chamber unit.
 5. The process as claimed in claim 1, wherein saidTALL-104 lymphocytes are genetically modified.
 6. The process as claimedin claim 1, wherein the complete culture medium in the multi-chamberstack amplification phase also comprises a maximum of 10% human serumand IL-2 in a concentration from 80 to 120 IU/ml.
 7. The process asclaimed in claim 6 wherein said complete culture medium comprises 4-6%human serum.
 8. A process according to claim 6, wherein said TALL-104lymphocytes are genetically modified.
 9. A process for the preparationof frozen bags of TALL-104 lymphocytes in an amount of at least 1×10⁹cells comprising: a. recovering at least 1×10⁹ TALL-104 cells grown in ahomogeneous culture system in a multi-chamber stack according to claim1; b. centrifuging the TALL-104 cells; c. collecting the TALL-104 cellsinto bags; and d. freezing the bags.
 10. The process as claimed in claim9, wherein the bags are sealed transversally to a bag filling collet atleast in two points to create at least a sampling chamber containing acell culture volume ranging from 0.1 to 1 ml, physically separated fromthe culture contained in the bag to perform quality controls.
 11. Aprocess for the preparation of a therapeutic dose of at least 1×10⁹TALL-104 lymphocytes in a homogeneous culture system comprising usingthe process according to claim
 1. 12. The process of claim 11, whereinthe complete culture medium in the multi-chamber stack amplificationphase also comprises a maximum of 10% human serum and IL-2 in aconcentration from 80 to 120 IU/ml.